Gene encoding b protein

ABSTRACT

An objective of this invention is to identify and provide a gene encoding medaka protein B. An additional objective of the present invention is to provide human and murine homologues of the gene. To achieve the above mentioned objectives, the inventors performed positional cloning on the B gene. The gene encoding medaka protein B was successfully isolated, and the present invention has come to completion. Accordingly, in accordance with the invention, there are provided a nucleic acid encoding medaka protein B and those encoding homologues thereof. According to another aspect of the invention, there are provided a nucleic acid listed in Seq. No. 1 that encodes medaka protein B and a nucleic acid listed in Seq. No. 3 that encodes mouse protein B/AIM-1. According to the invention, there is provided a method of suppressing melanin production characterized in that expression of the gene encoding protein B is suppressed.

TECHNICAL FIELD

[0001] The present invention relates to genes essential for melanin synthesis, particularly to a gene encoding protein B essential for melanin synthesis.

[0002] Throughout the following description, various publications will be referred to by numerical citation within parentheses. Full bibliographic citation of these publications will be found immediately before the sequence listing at the end of the Detailed Description.

BACKGROUND ART

[0003] For skin whitening as well as for prevention and treatment of skin spots and birthmarks associated with the formation of melanin pigment, conventionally, tyrosinase inhibitors have been used. However, there is still a strong demand for more efficacious medicaments and cosmetics that can be used safely for human as skin-whitening agents and therapeutic agents for skin spots and birthmarks. Accordingly, a method of suppressing melanin synthesis has been eagerly anticipated which is distinct from conventional ones.

[0004] Although melanin is known to be produced, in melanosomes within melanocytes, through tyrosinase-catalyzed and auto-oxidation of tyrosine, the mechanism of melanin production remains mostly unsolved, and most of the genes involved in melanin production have not been identified yet.

[0005] On the other hand, researches on vertebrate hair color have been pursued using primarily mouse as a model animal, and, during 1990s, a number of genes have been identified that are responsible for hair color mutation. These researches have not only made a great contribution to the elucidation of pigment cell differentiation at molecular level, also revealed their association with human genetic diseases (1-6). However, mammals, during the course of evolution, culminated in holding decreased kinds of pigment cells and losing mobility of pigment granules in cells. Therefore, it will be impossible to elucidate, by researches using mouse, the molecular mechanisms underlying the features recognized in lower vertebrates, namely, bright body coloration, marking formation, and rapid body color change for adaptive response to environment. In contrast, medaka (rice fish; Oryzias latipes) is thought to be equally qualified to zebrafish as a model organism of vertebrates, and recently bases for its molecular genetic researches have been rapidly being set (7, 8). Since medaka has all sets of pigment cells (black and yellow/red, silver, and white), and approximately 70 kinds of spontaneous mutants in body color have been separated and established as strains, it is thought to be very useful as a model organism for body color research (9). A medaka with orange color is called himedaka, which has been a popular companion animal in Japan from a long time ago. While xanthophore/erythrophore on body surface are normal in himedaka, the amount of melanin in its melanophore is extremely small. It is known that this phenotype is due to mutation of a single gene (B gene) located in a b locus. However, the B gene remains unidentified and its function is only presumptive.

DISCLOSURE OF INVENTION

[0006] Accordingly, an objective of the present invention is to identify and provide a gene encoding medaka protein B. An additional objective of the present invention is to provide human and murine homologues of the gene.

[0007] This invention also relates to a method of suppressing melanin production characterized in that expression of a nucleic acid(s) encoding medaka protein B or a homologue(s) thereof is(are) suppressed.

[0008] To achieve the above mentioned objectives, the inventors performed positional cloning on the B gene. The gene encoding medaka protein B was successfully isolated, and the present invention has come to completion.

[0009] Accordingly, in accordance with the invention, there are provided a nucleic acid encoding medaka protein B and those encoding homologues thereof.

[0010] According to another aspect of the invention, there are provided a nucleic acid listed in Seq. No. 1 that encodes medaka protein B and a nucleic acid listed in Seq. No. 3 that encodes mouse protein B/AIM-1.

[0011] In this case, the nucleic acids may be DNA, more preferably be cDNA or genomic DNA.

[0012] According to the invention, there is provided a method of suppressing melanin production characterized in that expression of the gene encoding protein B is suppressed.

BRIEF DESCRIPTION OF DRAWINGS

[0013]FIG. 1 is a photograph showing the difference between phenotypes of wild-type and b-locus mutants. From top to bottom: wild-type (B/B), b (b/b) and b^(g8) (b^(g8)/b^(g8))

[0014]FIG. 2 is a genetic and physical map of the b mutation candidate interval.

[0015] a, The recombination map of linkage group 12 (LG12).

[0016] b, Further recombination analysis with another 250 female meioses did not detect recombination between the b locus and OPH3-1. Terminal sequences of OPH3-1-positive BAC (171M23) were mapped and named 171M23F and 171M27R.

[0017] c, BAC171M23 was subcloned into cosmids and several OPH3-1-positive clones were isolated by PCR screening. Shown is one cosmid (C27) with an insert of about 40 kb, which was shown to contain the entire b-mutation candidate.

[0018] d, Shotgun sequencing determined 36.3 kb (>90%) of the C27 insert.

[0019]FIG. 3 shows medaka B cDNA and the deduced amino-acid sequence.

[0020] a, Nucleotide sequence of B and the amino-acid sequence deduced from its cDNA.

[0021] b, Sequence alignment of medaka protein B, human AIM-1 protein, and the mouse B/AIM-1 homologue.

[0022]FIG. 4 illustrates an experiment to investigate mutations of the B gene.

[0023] a, A gel-electrophoretic photograph after RT-PCR performed in two regions of B mRNA using pairs of primers indicated above.

[0024] b, Model for the arrangement of the B polypeptide in the membrane.

[0025]FIG. 5 illustrates an experiment to investigate expression of the B gene.

[0026] a, A gel-electrophoretic photograph after RT-PCR was performed independently on two regions of B mRNA using a primer pair indicated at the top of the figure, and then expression was continuously detected from the day of fertilization (day 0) until hatching (day 7).

[0027] b, A microphotograph showing the in situ hybridization with the partial B cDNA riboprobes to a two-day embryo of the albino i6 strain which completely lacks melanin in the melanophores. Left: sense probe, right; antisense probe.

[0028] c, A microphotograph showing distribution of melanophores in a wild-type embryo.

[0029] d, A microphotograph showing expression of B in two-day embryos of the b strain. Left: sense probe, right; antisense probe.

[0030] e, A gel-electrophoretic photograph showing B gene expression in adult organs. The primers indicated by black arrows in FIG. 5a were used.

[0031] In FIG. 6 is shown an amino acid sequence alignment of medaka protein B (HNI strain) and Apium graveolens sucrose transporter (agSUT1).

BEST MODE FOR CARRYING OUT OF THE INVENTION

[0032] Hereafter, the invention will be illustrated in more detail.

[0033] The inventors have identified the medaka B gene using positional cloning.

[0034] The positional cloning is an approach in which the position of a responsible gene on a chromosome is defined with a variety of methods to carry on the cloning of the responsible gene with the aid of the defined position. The position and region of a target gene are determined by means of linkage analysis method using crossing. Molecular (DNA) markers are essential for these analyses. Physical mappings are made by constructing, with the use of chromosome walking using the above markers and BAC (bacterial artificial chromosome) and the like, a contig over a region where the target gene is located. Confirmation of a candidate gene will be achieved by capturing gene fragments expressed in the above regions to analyze DNA polymorphism between normal individuals and mutants. Screening of full-length cDNA clones thereafter, for example, will be performed conventionally with general cloning techniques using the above gene fragments as probes.

[0035] Specifically, the inventors isolated the B gene as a responsible gene for a mutant in b locus by means of linkage analysis using crossing between wild-type and himedaka strains, and chromosome walking using BAC. In this BAC DNA stage, the target region can be narrowed down to 200 Mb in size. Then techniques will be applied in which the cloned DNA in BAC is fragmented and subcloned into a vector using E. coli as a host. It is most preferable to subclone DNA fragments digested to 30-40 kb into a cosmid vector. Therefore, in this study, cosmid libraries were prepared from BAC containing the b locus. Screening was carried on to narrow the candidate region to less than 40 kb. As a result of sequencing an insert in a cosmid containing the candidate region for b mutation, a candidate gene having high homology with human MACR1 and AIM-1 was found. While neither mutation of nucleic acids nor expression of abnormal mRNAs was detected in medaka MACR1, a mutation was identified in AIM-1 homologous gene, and the inventors designated the gene as B.

[0036] The nucleic acid sequence encoding medaka protein B (Seq. No. 4) is that listed in Seq. No. 1. A human homologue of medaka protein B encodes human AIM-1 (Seq. No. 5), which is reported as an EST isolated from melanoma cells. The nucleotide sequence encoding human AIM-1 is that listed in Seq. No. 2. The nucleotide sequence encoding a murine homologue of medaka protein B (Seq. No. 6) is that listed in Seq. No. 3 (FIG. 3). The murine homologue according to the invention was cloned with degenerated primers. Namely, cDNAs for cloning the murine homologue of the invention can be prepared by synthesizing single-stranded cDNAs, with the use of a commercial kit using mRNAs from murine cells as templates, by means of oligo dT primers and reverse transcriptase. In order to isolate the DNA of interest with the cDNAs prepared as described above, for example, degenerate primers are first synthesized that are corresponding to the amino acid sequence of medaka protein B, then degenerate polymerase chain reaction (also referred to as degenerate PCR) is carried out. Primers are then synthesized based on the resultant partial sequence from gene of interest, and 5′ and 3′ RACE are performed using the primers. “RACE” (rapid amplification of cDNA ends) herein mentioned is a method of running PCR based on nucleotide sequence information obtained from a known region, when the nucleotide sequence of cDNA is previously known in part, to clone an unknown region up to cDNA ends. When the unknown region is located upstream (5′) side of cDNA it is called 5′ RACE, and when located downstream (3′) it is called 3′ RACE. For example, in a case of 5′ RACE, antisense primers are synthesized on the basis of a known nucleotide sequence to make a reverse transcription reaction using the synthesized primers. Subsequent digestion of mRNA (forms a double strand with cDNA) with RNaseH results in single-stranded first-strand cDNA. While the first-strand cDNA contains a primer-derived sequence in its 5′ terminus, the 3′ terminus ends with an unknown sequence. Therefore, an anchor sequence is to be added to the 3′ terminus in order to amplify the unknown sequence by PCR. The cDNA having an unknown region in its 5′ upstream region can be amplified by running PCR, after the addition of the anchor sequence, using a nucleotide primer complementary to the anchor sequence and an antisense primer specific for a partial region whose sequence is known. In addition to cDNA, genomic DNA may be included as a template DNA used for degenerate PCR.

[0037] Nucleotide sequences of DNA fragments prepared with 5′ RACE and 3′ RACE are then determined. The nucleotide sequences can be determined by conventional methods such as chemical modification method of Maxam-Gilbert or dideoxynucleotide chain-termination method using M13 phage. Generally, automatic nucleotide sequencers (e.g., 373A DNA sequencer manufactured by PERKIN-ELMER Co. Ltd.) will be used for sequencing.

[0038] The function of the medaka protein B or homologues thereof described above has not been known so far, and they have never been reported to have similar characteristics to those found in the present invention. Also, it has not been reported that the genes of the present invention are essential for melanin synthesis.

[0039] The nucleic acids of the invention can be produced by the methods described above and those described in the Examples. Since their nucleotide sequences have already been determined, they can also be produced by chemical synthesis, by PCR using the genes as templates, or by hybridization using DNA fragments sharing the nucleotide sequences of the genes as probes. Designed primers or probes can be chemically synthesized according to their nucleotide sequences. PCR can be performed according to conventional procedures.

[0040] As used herein, the term “medaka protein B or homologues thereof” refers to full-length medaka protein B or homologues thereof, biologically active fragments thereof, or functional equivalents of the medaka protein B or homologues thereof. As functional equivalents of the medaka protein B or homologues thereof, modified medaka protein Bs or homologues thereof having any biological activity may be included. Functional equivalents of the medaka protein B or homologues thereof may include, for example, those proteins wherein one or several amino acid(s) is (are) replaced for (e.g. replacement of one acidic amino acid with a different acidic amino acid), deleted from, or added to their amino acid sequences.

[0041] The nucleic acids of the invention encoding the medaka protein B or homologues thereof may encode either whole of these proteins or shorter peptide sequences that can have an effect on melanin synthesis. Alternatively, said nucleic acids may have nucleic acid sequences that encode, due to degeneracy in genetic codes, the gene products identical to the medaka protein B or homologues thereof.

[0042] As an example of the nucleic acid sequences of the invention encoding the medaka protein B or homologues thereof, the followings can beincluded: DNA sequences encoding the medaka protein B or homologues thereof, and RNAs encoded by the DNA sequences.

[0043] As used herein, the term “homologues of the medaka protein B” means proteins having homology to the medaka protein B. More specifically, they share preferably 45% or more, more preferably 60% or more, more preferably 75% or more, even more preferably 90% or more, and most preferably 95% or more sequence homology with the deduced amino acid sequence of the medaka protein B.

[0044] In order to search the homologues described above and their homology, sequences available in public databases can be used for searching with the aid of program (e.g., Blastn/Blastx25, but not limited to these).

[0045] By comparison with the amino acid sequences described above, the medaka protein B will be found to share 55% amino acid identity with human AIM-1 (see FIG. 3).

[0046] General procedures can be used to suppress the expression of nucleic acids. For example, antisense method may be used. In this method, antisense RNA is injected exogenously into adult animals or cells; wherein the antisense RNA is such RNA containing a sequence complementary to whole or part of an mRNA nucleotide sequence. The RNA so injected inhibits, through the formation of a hybrid with the mRNA, a process of translation of genetic information carried by mRNA into protein. Said antisense RNA can be expressed within the cells as antisense RNA by incorporating its DNA information into an expression vector. Since they both are structurally complementary to target mRNA, they can bind to the target mRNA to suppress the expression of target gene. For exhibiting antisense effect, the antisense RNA may not be 100% complementary to the target RNA. The complementarity may not be high so long as the expression of the protein of the invention can be suppressed. The antisense RNA shares preferably 90%, more preferably 95% complementarity with the RNA of the invention. For exhibiting antisense effect, the length of the complementary RNA must be at least 15 bp, preferably 100 bp or more, more preferably 500 bp or more. We can also use antisense DNA complementary to the DNA encoding the medaka protein B or a part thereof. The antisense RNA complementary to the DNA of the invention can be prepared according to general procedures.

EXAMPLES

[0047] This invention will be illustrated in more detail with reference to the following Examples. However, one skilled in the art will readily appreciate that these Examples are merely illustrative of the invention and should not be intrigued as limiting the scopes of the invention.

[0048] Methods

[0049] Linkage Analysis of Medaka;

[0050] Linkage analysis was performed using backcross progeny from genetically divergent HNI (+/+) (12) and AA2 (b/b) (13) inbred strains. The BAC genomic library used for screening is based on the Hd-rR (b/b) inbred strain (8).

[0051] Genomics;

[0052] BAC and cosmid ends were sequenced directly after purifying BAC or cosmid DNA using the Plasmid Maxi Kit (Qiagen), and mapped as described elsewhere (11). BAC171M23 was subcloned into the SuperCos 1 Cosmid Vector after Sau3AI partial digestion, followed by phage packaging using MaxPlax Lambda Packaging Extract (Epicenter Technologies). A shotgun library was constructed by subcloning Sau3AI-partially-digested C27 fragments into BamHI site of pUC118. Inserts were amplified by colony PCR and sequenced on the Applied Biosystems model 377 automated DNA sequencer. Sequence data were manually assembled using Genetixmac. The C27 sequence was compared with sequences that are publicly available in databases, using the Blastn and Blastx (25) programs.

[0053] Mutation Analyses;

[0054] First-strand cDNA was prepared from total RNA isolated from hatched fries using Isogen (Nippon Gene). RT-PCR of the b alleles was performed in three or more overlapping regions using the following parameters: 30 cycles of 98° C. for 20s, 60° C. for 1 min, and 72° C. for 2 min, and products were sequenced directly.

[0055] Mouse B/AIM-1 Homologue;

[0056] Nested degenerate primers were designed from medaka B and human AIM-1 sequences, and mouse cDNA from adult (albino) eyes, kidneys, and uterus were used as templates for RT-PCR. To determine the whole sequence of the ORF, specific primers were designed to the amplified sequence for 3′-RACE and 5′-RACE.

[0057] Expression;

[0058] Fertilized eggs of the Sakura strain (+/+) were incubated at 25° C. and killed 0-7 days after fertilization. To assess adult expression, the HNI strain was used. RT-PCR products were separated electrophoretically on 1% agarose gel and examined under UV-transillumination after ethidium bromide staining. Whole mount in situ hybridization was performed by hybridizing DIG-labelled partial B riboprobes to tyrosinase deletion mutant (i6/i6) (26) embryos.

[0059] Results

[0060] We have previously reported an STS (called OPH3-1) that is tightly linked to the b locus (11) (FIG. 2a). Further recombination analysis mapped the b locus to within 0-0.7 cM (0/545) of OPH3-1. Because the two inbred strains used for recombination mapping, HNI (12) (+/+) and AA2 (13) (b/b), are extremely polymorphic, it is not difficult to detect SNPs and/or other polymorphisms in every 100 bp of genomic sequence. To take advantage of this, we detected polymorphisms in terminal sequences of OPH3-1-positive BACs and mapped them using a PCR/PCR-RFLP method. Because an unexpectedly high level of recombination was detected (nine recombination within one BAC) (FIG. 2b), we further subcloned the BAC (171M23) into cosmids and isolated a cosmid (C27) that contains the entire b-mutation candidate region (FIG. 2c).

[0061] In this study, the extreme polymorphism and high recombination frequency enabled us to narrow the b-mutation candidate region to less than 40 kb by analyzing only 545 backcross progeny. This intriguingly high recombination frequency around the b locus is a female-meiosis-specific phenomenon, and it is about 10 times less frequent in males.

[0062] The BAC library screened was based on the Hd-rR (b/b) inbred strain (8). However, traces of B sequence were likely to exist in C27, because b is not a phenotypically null allele (FIG. 1). We actually obtained two B-candidate genes from 36.3 kb (>90%) of the sequence of the C27 insert (FIG. 2d). These were highly homologous to human genes encoding alpha-methylacyl-CoA racemase (MACR1) and AIM-1 protein. MACR1 catalyses the interconversion of R- and S-stereoisomers of alpha-methyl-branched-chain fatty acyl-CoA esters, and mutations in it cause adult-onset sensory motor neuropathy (14). AIM-1 has been reported as an EST isolated from melanocytes and melanoma cells and, more recently, as a new antigen on HLA-A2-expressing melanoma cells recognized by T cells (15).

[0063] Although neither nucleotide mutations nor abnormal mRNA expression was detected for medaka MACR1, we identified mutations on medaka AIM-1 in seven of eight b-locus mutants examined and named the gene B.

[0064] The whole B ORF was sequenced by 3′-RACE and its exon-intron boundaries determined (FIG. 3a). The deduced B protein consists of 576 amino acids and is 55% identical (304/543) to human AIM-1, although its N-terminus and middle regions are less conserved (FIG. 3b). Hydropathy analysis indicated that both medaka protein B and human AIM-1, and the mouse B/AIM-1 homologue which we isolated by degenerate RT-PCR, have 12 hydrophobic transmembrane domains and a less-conserved long loop between the sixth and seventh transmembrane domains (FIG. 3b).

[0065] We analyzed two spontaneous (b and b^(p)), one radiation-induced (b^(g8)), and five ENU-induced (b^(g21), b^(d2), b^(d4), b^(d8), and b^(c1)) mutants. Abnormally longer RT-PCR products were amplified from b^(p) and b^(g8) (FIG. 4a). The b^(p) transcript has an insertion of 195 nucleotides, which is identical to the adjacent ORF sequence. This tandem repeat produces an insertion of 65 amino acids, which disrupts the normal conformation and function of B. The b^(g8) transcript has an insert of 699 nucleotides due to a missplicing of the last intron caused by a two-base deletion at the splice-donor consensus sequence. A premature stop codon in the intron causes truncation of B protein and loss of the eleventh and twelfth transmembrane domains. The b^(c1) transcript is normal in size but has a nonsense point mutation changing GAG (glutamine) to TAG, resulting in the loss of six C-terminus transmembrane domains. These results are consistent with phenotypic observation that these three more severe mutations result in less-melanized melanophores than do the b^(g21), b^(d4), b^(d2), and b^(d8) missense point mutations that introduce charged hydrophilic amino acids into the transmembrane domains (FIG. 4b). In the b allele, the mutation does not occur in the ORF. However, our recombination map clearly shows that the b mutation is within the region covered by C27 that contains the first 1035 bp of the B ORF (FIG. 2c and d). Because b is distinguished from the other alleles in manifesting tissue-specific defects (FIG. 1) and B gene expression is disrupted in the b in

[0066] a tissue-specific manner, the b mutation may be in a gene-regulating region contained in C27:

[0067] a promoter, an enhancer, the 5′-UTR, or an intron.

[0068] The B gene is continuously expressed throughout embryonic development at levels detectable by RT-PCR (FIG. 5a). B transcripts detected at the blastula stage (day0) may be maternal. Strong expression signals were observed on the eyes, body, and yolk sac of two-day embryos (FIG. 5b), where melanophores are distributed in wild-type embryos (FIG. 5c). The signals on the body and yolk sac disappeared as the embryos developed, but those on the eyes were detected continuously until hatching, indicating transient B expression in developing melanophores. Adult fish also express the B gene and its expression is not restricted to the melanophore lineage (FIG. 5e). On the other hand, expression of human AIM-1 was not detected in any tissue other than melanocytes and melanoma cells by northern hybridization (15). However, almost identical ESTs from the kidney and uterus are reported in the databases. Furthermore, we isolated the mouse B/AIM-1 homologue from the eyes as well as from the kidney and uterus by degenerate RT-PCR. Therefore, B/AIM-1 genes are likely to be expressed in several organs although their strongest expression is restricted to the melanophore lineage.

[0069] With identification of the medaka B gene, we can begin to understand how this gene affects vertebrate melanization. Because the b mutation defect is manifested inside the melanosome (10) and many twelve-transmembrane proteins have been known to function as transporters (16-18), we propose that the B protein may be a component of the melanosomal membrane, transporting certain substances required for melanin biosynthesis. In this regard, it is quite informative that B has <23% (115/490) amino-acid identity with and structural similarity to plant sucrose-proton symporters (19) (FIG. 6). In particular, the region between the second and third transmembrane domains has significant similarity, where a completely conserved putative motif appears in all plant sequences known to function as sucrose transporters (19), in medaka B, in human AIM-1 and its mouse homologue, and also in the Drosophila CG4484 gene product (29% identical to medaka B). Although an effect of sucrose on melanin synthesis has not been postulated, mouse B16 melanoma cells cultured in medium containing galactose have much higher tyrosinase activity and produce more melanin than cells in medium containing glucose (20-21). Furthermore, it was recently reported that mutations at tyrosinase N-glycosylation sites reduce tyrosinase activity (22), indicating an effect of certain saccharides on melanin biosynthesis. Detailed functional analysis of the B/AIM-1 protein may provide new insights into the biochemical regulation of melanin synthesis.

Advantage of the Invention

[0070] Through the use of the gene according to the present invention, a method will be achieved for suppressing melanin production in a manner different from conventional methods.

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1 6 1 1979 DNA Oryzias latipes CDS (184)..(1911) 1 cttccttctg tgaagttatg cctagtttct ggactctttg ctcagctatg caataagaaa 60 gaggattaac ggagccgagc agcaggaatt tggcctttca tccatcacct ggttccctta 120 aagaaaatac tcttcctctc tttcctttct ttctttcttt ctttcttcta ccctctttcc 180 agc atg acc ctg ctg tca gag gac cag tct gcc agg ccc cag ccc tgt 228 Met Thr Leu Leu Ser Glu Asp Gln Ser Ala Arg Pro Gln Pro Cys 1 5 10 15 ctg ctg tca gaa cct gac aaa cac atc agc act tct tta gac cag tac 276 Leu Leu Ser Glu Pro Asp Lys His Ile Ser Thr Ser Leu Asp Gln Tyr 20 25 30 aga cca gac tac act aaa gag gac tac atg gac agt gtg gag acc tca 324 Arg Pro Asp Tyr Thr Lys Glu Asp Tyr Met Asp Ser Val Glu Thr Ser 35 40 45 gtg ttt ggg act gtg gag ccc cct cga cgt tct cgt ggt cgc ctg atc 372 Val Phe Gly Thr Val Glu Pro Pro Arg Arg Ser Arg Gly Arg Leu Ile 50 55 60 ctc cac agt atg gtt atg ttt ggt cgg gag ttc tgc tat gcg gtg gaa 420 Leu His Ser Met Val Met Phe Gly Arg Glu Phe Cys Tyr Ala Val Glu 65 70 75 gcg gca ttt gtc aca ccg gtg ctt ctc agc gta ggt ctc ccc cgc agt 468 Ala Ala Phe Val Thr Pro Val Leu Leu Ser Val Gly Leu Pro Arg Ser 80 85 90 95 ctc tac agt ctg gtg tgg ctg atc agc ccc atc ctg ggc ttt ctg ctt 516 Leu Tyr Ser Leu Val Trp Leu Ile Ser Pro Ile Leu Gly Phe Leu Leu 100 105 110 cag ccc atc atc ggc tca gca agt gac tac tgc cgg tct tcc tgg gga 564 Gln Pro Ile Ile Gly Ser Ala Ser Asp Tyr Cys Arg Ser Ser Trp Gly 115 120 125 agg agg agg ccc tac atc ctt gtc ctc ggt atc ctc atg ctg gtg ggg 612 Arg Arg Arg Pro Tyr Ile Leu Val Leu Gly Ile Leu Met Leu Val Gly 130 135 140 ctc agc atg ttt ctc aat gga gat gca gtg gtc tca gag ctc gtc agt 660 Leu Ser Met Phe Leu Asn Gly Asp Ala Val Val Ser Glu Leu Val Ser 145 150 155 gac agg tca tcc aga agt acg tgg gcc atc gtg gtg gtc atg ttt ggg 708 Asp Arg Ser Ser Arg Ser Thr Trp Ala Ile Val Val Val Met Phe Gly 160 165 170 175 gtt gtg ctg ttc gac ttt gct gca gat ttc atc gac ggg cca atc aag 756 Val Val Leu Phe Asp Phe Ala Ala Asp Phe Ile Asp Gly Pro Ile Lys 180 185 190 gcc tac ctg ttt gat gtg tgc tca tac caa gac aag gaa aga ggt ctt 804 Ala Tyr Leu Phe Asp Val Cys Ser Tyr Gln Asp Lys Glu Arg Gly Leu 195 200 205 cat tat cac gct tta ttc aca gga ctg ggc gga gct tgt ggt tac ctg 852 His Tyr His Ala Leu Phe Thr Gly Leu Gly Gly Ala Cys Gly Tyr Leu 210 215 220 gtt gga gca atg gac tgg ggt cac tct gta ttg ggg cgc ctc ctg ggc 900 Val Gly Ala Met Asp Trp Gly His Ser Val Leu Gly Arg Leu Leu Gly 225 230 235 tct gag tac caa gtg atc tac ttc ttc tct gca ctc act tgg ggc gtc 948 Ser Glu Tyr Gln Val Ile Tyr Phe Phe Ser Ala Leu Thr Trp Gly Val 240 245 250 255 ttt ctc atc gtg cac ctc ttc agt att cca gag aaa cct ctg gcc aaa 996 Phe Leu Ile Val His Leu Phe Ser Ile Pro Glu Lys Pro Leu Ala Lys 260 265 270 gtg cca tct gaa tcc tca gct tcc agt gcg ctc cgt tta cta ggc ccc 1044 Val Pro Ser Glu Ser Ser Ala Ser Ser Ala Leu Arg Leu Leu Gly Pro 275 280 285 cac agc aat ggc tac gga gca ctg ggg aag gag cct gtc tct cct gtc 1092 His Ser Asn Gly Tyr Gly Ala Leu Gly Lys Glu Pro Val Ser Pro Val 290 295 300 atc cca aca tcc agc ccc gag atc agg cca agg tcc tac tcg gcg ctt 1140 Ile Pro Thr Ser Ser Pro Glu Ile Arg Pro Arg Ser Tyr Ser Ala Leu 305 310 315 gga gaa aga ccc aga tct ttc tct gct ctc ggt gaa gcc aac tct gtg 1188 Gly Glu Arg Pro Arg Ser Phe Ser Ala Leu Gly Glu Ala Asn Ser Val 320 325 330 335 act tca agt gcc aaa cag cca att aag gag gac cag aag aag atg acg 1236 Thr Ser Ser Ala Lys Gln Pro Ile Lys Glu Asp Gln Lys Lys Met Thr 340 345 350 ttc agg tct ttg atg aaa gct atc ttc aac atg cca aac cac tac cgc 1284 Phe Arg Ser Leu Met Lys Ala Ile Phe Asn Met Pro Asn His Tyr Arg 355 360 365 ttc ctg tgc atc agc cac ctg ctg gga tgg gca gct ttt ctc tgc aac 1332 Phe Leu Cys Ile Ser His Leu Leu Gly Trp Ala Ala Phe Leu Cys Asn 370 375 380 atg ctc ttc ttc act gac ttc atg gga cag att gtt tat agg gga aat 1380 Met Leu Phe Phe Thr Asp Phe Met Gly Gln Ile Val Tyr Arg Gly Asn 385 390 395 cct tat gca gaa cac aac tcc acg gct tac atc acg tat gag aga gga 1428 Pro Tyr Ala Glu His Asn Ser Thr Ala Tyr Ile Thr Tyr Glu Arg Gly 400 405 410 415 gtt gaa gtt ggc tgc tgg gga ctg tgt att aat gca gtt tcc tct gct 1476 Val Glu Val Gly Cys Trp Gly Leu Cys Ile Asn Ala Val Ser Ser Ala 420 425 430 ctc tac tcg tac gtg cag cgc ttc ctc ctt cca tac att ggc ctg aag 1524 Leu Tyr Ser Tyr Val Gln Arg Phe Leu Leu Pro Tyr Ile Gly Leu Lys 435 440 445 gga tta tac ttc atg ggc tac ttt gtg ttc ggc atg ggt acc agt cta 1572 Gly Leu Tyr Phe Met Gly Tyr Phe Val Phe Gly Met Gly Thr Ser Leu 450 455 460 att ggc ctc ttc cca gaa gtc ata gcc acg ctc atc ctc tgc agc gtg 1620 Ile Gly Leu Phe Pro Glu Val Ile Ala Thr Leu Ile Leu Cys Ser Val 465 470 475 ttt ggc gtc atg tct agc acg ctc tac acg atc ccg ttc aac cta att 1668 Phe Gly Val Met Ser Ser Thr Leu Tyr Thr Ile Pro Phe Asn Leu Ile 480 485 490 495 gct gag tat cag cgg gag gaa gag gaa caa gta aag ctg gag gga ggc 1716 Ala Glu Tyr Gln Arg Glu Glu Glu Glu Gln Val Lys Leu Glu Gly Gly 500 505 510 aat gaa agc cct cga ggc acc ggg atg gac tgt gct gcg ctc act tgt 1764 Asn Glu Ser Pro Arg Gly Thr Gly Met Asp Cys Ala Ala Leu Thr Cys 515 520 525 atg gtc cag ctg gct caa atc att gtg ggg gcg gga ctc ggg gct ctg 1812 Met Val Gln Leu Ala Gln Ile Ile Val Gly Ala Gly Leu Gly Ala Leu 530 535 540 gtc aac gcg gca gga agt gtc atc gtg gtg gtt cta tca gct tcc tct 1860 Val Asn Ala Ala Gly Ser Val Ile Val Val Val Leu Ser Ala Ser Ser 545 550 555 atc tcc ctc ata ggc tgc atc ttc att gct ctc ttt atc aga tat gtg 1908 Ile Ser Leu Ile Gly Cys Ile Phe Ile Ala Leu Phe Ile Arg Tyr Val 560 565 570 575 gaa tgatatttct tttaaagtga actgtgctgc tttattaaat aaagagtctt 1961 Glu taatatatca aaaaaaaa 1979 2 576 PRT Oryzias latipes 2 Met Thr Leu Leu Ser Glu Asp Gln Ser Ala Arg Pro Gln Pro Cys Leu 1 5 10 15 Leu Ser Glu Pro Asp Lys His Ile Ser Thr Ser Leu Asp Gln Tyr Arg 20 25 30 Pro Asp Tyr Thr Lys Glu Asp Tyr Met Asp Ser Val Glu Thr Ser Val 35 40 45 Phe Gly Thr Val Glu Pro Pro Arg Arg Ser Arg Gly Arg Leu Ile Leu 50 55 60 His Ser Met Val Met Phe Gly Arg Glu Phe Cys Tyr Ala Val Glu Ala 65 70 75 80 Ala Phe Val Thr Pro Val Leu Leu Ser Val Gly Leu Pro Arg Ser Leu 85 90 95 Tyr Ser Leu Val Trp Leu Ile Ser Pro Ile Leu Gly Phe Leu Leu Gln 100 105 110 Pro Ile Ile Gly Ser Ala Ser Asp Tyr Cys Arg Ser Ser Trp Gly Arg 115 120 125 Arg Arg Pro Tyr Ile Leu Val Leu Gly Ile Leu Met Leu Val Gly Leu 130 135 140 Ser Met Phe Leu Asn Gly Asp Ala Val Val Ser Glu Leu Val Ser Asp 145 150 155 160 Arg Ser Ser Arg Ser Thr Trp Ala Ile Val Val Val Met Phe Gly Val 165 170 175 Val Leu Phe Asp Phe Ala Ala Asp Phe Ile Asp Gly Pro Ile Lys Ala 180 185 190 Tyr Leu Phe Asp Val Cys Ser Tyr Gln Asp Lys Glu Arg Gly Leu His 195 200 205 Tyr His Ala Leu Phe Thr Gly Leu Gly Gly Ala Cys Gly Tyr Leu Val 210 215 220 Gly Ala Met Asp Trp Gly His Ser Val Leu Gly Arg Leu Leu Gly Ser 225 230 235 240 Glu Tyr Gln Val Ile Tyr Phe Phe Ser Ala Leu Thr Trp Gly Val Phe 245 250 255 Leu Ile Val His Leu Phe Ser Ile Pro Glu Lys Pro Leu Ala Lys Val 260 265 270 Pro Ser Glu Ser Ser Ala Ser Ser Ala Leu Arg Leu Leu Gly Pro His 275 280 285 Ser Asn Gly Tyr Gly Ala Leu Gly Lys Glu Pro Val Ser Pro Val Ile 290 295 300 Pro Thr Ser Ser Pro Glu Ile Arg Pro Arg Ser Tyr Ser Ala Leu Gly 305 310 315 320 Glu Arg Pro Arg Ser Phe Ser Ala Leu Gly Glu Ala Asn Ser Val Thr 325 330 335 Ser Ser Ala Lys Gln Pro Ile Lys Glu Asp Gln Lys Lys Met Thr Phe 340 345 350 Arg Ser Leu Met Lys Ala Ile Phe Asn Met Pro Asn His Tyr Arg Phe 355 360 365 Leu Cys Ile Ser His Leu Leu Gly Trp Ala Ala Phe Leu Cys Asn Met 370 375 380 Leu Phe Phe Thr Asp Phe Met Gly Gln Ile Val Tyr Arg Gly Asn Pro 385 390 395 400 Tyr Ala Glu His Asn Ser Thr Ala Tyr Ile Thr Tyr Glu Arg Gly Val 405 410 415 Glu Val Gly Cys Trp Gly Leu Cys Ile Asn Ala Val Ser Ser Ala Leu 420 425 430 Tyr Ser Tyr Val Gln Arg Phe Leu Leu Pro Tyr Ile Gly Leu Lys Gly 435 440 445 Leu Tyr Phe Met Gly Tyr Phe Val Phe Gly Met Gly Thr Ser Leu Ile 450 455 460 Gly Leu Phe Pro Glu Val Ile Ala Thr Leu Ile Leu Cys Ser Val Phe 465 470 475 480 Gly Val Met Ser Ser Thr Leu Tyr Thr Ile Pro Phe Asn Leu Ile Ala 485 490 495 Glu Tyr Gln Arg Glu Glu Glu Glu Gln Val Lys Leu Glu Gly Gly Asn 500 505 510 Glu Ser Pro Arg Gly Thr Gly Met Asp Cys Ala Ala Leu Thr Cys Met 515 520 525 Val Gln Leu Ala Gln Ile Ile Val Gly Ala Gly Leu Gly Ala Leu Val 530 535 540 Asn Ala Ala Gly Ser Val Ile Val Val Val Leu Ser Ala Ser Ser Ile 545 550 555 560 Ser Leu Ile Gly Cys Ile Phe Ile Ala Leu Phe Ile Arg Tyr Val Glu 565 570 575 3 1650 DNA Homo sapiens CDS (26)..(1615) 3 caggaaggtt cctctcccag tggcc atg ggt agc aac agt ggg cag gct ggc 52 Met Gly Ser Asn Ser Gly Gln Ala Gly 1 5 cgc cac atc tat aaa tcc cta gct gat gat ggc ccc ttt gac tct gtg 100 Arg His Ile Tyr Lys Ser Leu Ala Asp Asp Gly Pro Phe Asp Ser Val 10 15 20 25 gag ccg cct aaa aga ccc acc agc aga ctc atc atg cac agc atg gcc 148 Glu Pro Pro Lys Arg Pro Thr Ser Arg Leu Ile Met His Ser Met Ala 30 35 40 atg ttc gga aga gag ttc tgc tac gcg gtg gag gca gcg tat gtg acc 196 Met Phe Gly Arg Glu Phe Cys Tyr Ala Val Glu Ala Ala Tyr Val Thr 45 50 55 cca gtc ctg ctc agc gta ggt ctg ccc agc agc ctg tac agc att gtg 244 Pro Val Leu Leu Ser Val Gly Leu Pro Ser Ser Leu Tyr Ser Ile Val 60 65 70 tgg ttc ctc agc ccc atc ctg gga ttc ctg ctg cag ccc gtg gtc gga 292 Trp Phe Leu Ser Pro Ile Leu Gly Phe Leu Leu Gln Pro Val Val Gly 75 80 85 tcg gcc agc gac cac tgc cgg tcc agg tgg ggc cgc cgg aga ccc tac 340 Ser Ala Ser Asp His Cys Arg Ser Arg Trp Gly Arg Arg Arg Pro Tyr 90 95 100 105 atc ctc acc ctg gga gtc atg atg ctc gtg ggc atg gct ctg tac ctc 388 Ile Leu Thr Leu Gly Val Met Met Leu Val Gly Met Ala Leu Tyr Leu 110 115 120 aat ggg gct act gtt gta gca gct ttg att gct aac cca agg agg aag 436 Asn Gly Ala Thr Val Val Ala Ala Leu Ile Ala Asn Pro Arg Arg Lys 125 130 135 ctg gtt tgg gcc ata agt gtc acc atg ata ggt gtc gtt ctc ttt gat 484 Leu Val Trp Ala Ile Ser Val Thr Met Ile Gly Val Val Leu Phe Asp 140 145 150 ttt gct gcc gac ttc att gat ggg ccc atc aaa gcc tac tta ttt gat 532 Phe Ala Ala Asp Phe Ile Asp Gly Pro Ile Lys Ala Tyr Leu Phe Asp 155 160 165 gtc tgc tcc cat cag gac aag gag aag ggc ctc cac tac cat gcc ctc 580 Val Cys Ser His Gln Asp Lys Glu Lys Gly Leu His Tyr His Ala Leu 170 175 180 185 ttc aca ggt ttt gga ggt gcc ctg ggt tac ctt ttg ggt gct ata gac 628 Phe Thr Gly Phe Gly Gly Ala Leu Gly Tyr Leu Leu Gly Ala Ile Asp 190 195 200 tgg gcc cat ctg gag ctg gga aga ctg ttg ggt aca gaa ttc cag gtc 676 Trp Ala His Leu Glu Leu Gly Arg Leu Leu Gly Thr Glu Phe Gln Val 205 210 215 atg ttc ttc ttc tct gca ttg gtg ctc act ttg tgt ttt act gtt cat 724 Met Phe Phe Phe Ser Ala Leu Val Leu Thr Leu Cys Phe Thr Val His 220 225 230 ctg tgc agt atc tct gaa gcc cca ctt aca gag gtt gca aag ggc att 772 Leu Cys Ser Ile Ser Glu Ala Pro Leu Thr Glu Val Ala Lys Gly Ile 235 240 245 ccc cca cag caa acc cct cag gac cct cca ttg tca tca gat gga atg 820 Pro Pro Gln Gln Thr Pro Gln Asp Pro Pro Leu Ser Ser Asp Gly Met 250 255 260 265 tac gag tat ggt tct atc gag aaa gtt aaa aat ggt tac gta aat cca 868 Tyr Glu Tyr Gly Ser Ile Glu Lys Val Lys Asn Gly Tyr Val Asn Pro 270 275 280 gag ctg gca atg cag gga gca aaa aac aaa aat cat gct gaa cag act 916 Glu Leu Ala Met Gln Gly Ala Lys Asn Lys Asn His Ala Glu Gln Thr 285 290 295 cgc agg gca atg aca tta aag tca ctg ctg aga gca ctg gtg aac atg 964 Arg Arg Ala Met Thr Leu Lys Ser Leu Leu Arg Ala Leu Val Asn Met 300 305 310 cct cct cac tac cgc tac ctt tgc atc agc cac ctc att gga tgg acg 1012 Pro Pro His Tyr Arg Tyr Leu Cys Ile Ser His Leu Ile Gly Trp Thr 315 320 325 gcc ttc ctg tcc aac atg ctg ttc ttc aca gat ttc atg ggc cag att 1060 Ala Phe Leu Ser Asn Met Leu Phe Phe Thr Asp Phe Met Gly Gln Ile 330 335 340 345 gtg tac cgc ggg gat ccc tat agt gca cac aac tcc aca gag ttt ctc 1108 Val Tyr Arg Gly Asp Pro Tyr Ser Ala His Asn Ser Thr Glu Phe Leu 350 355 360 atc tac gaa aga gga gtc gag gtt gga tgt tgg ggc ttc tgc atc aac 1156 Ile Tyr Glu Arg Gly Val Glu Val Gly Cys Trp Gly Phe Cys Ile Asn 365 370 375 tcc gtg ttt tcc tca ctt tat tct tac ttt cag aaa gtt ttg gta tcc 1204 Ser Val Phe Ser Ser Leu Tyr Ser Tyr Phe Gln Lys Val Leu Val Ser 380 385 390 tac att gga tta aag ggt ctt tac ttc acg gga tat ttg ctg ttt ggc 1252 Tyr Ile Gly Leu Lys Gly Leu Tyr Phe Thr Gly Tyr Leu Leu Phe Gly 395 400 405 ctg ggg acg gga ttt att ggg ctc ttc ccg aat gtc tac tcc acc ctg 1300 Leu Gly Thr Gly Phe Ile Gly Leu Phe Pro Asn Val Tyr Ser Thr Leu 410 415 420 425 gtc ctg tgc agc ctg ttt ggt gta atg tcc agc acc ctg tac act gtg 1348 Val Leu Cys Ser Leu Phe Gly Val Met Ser Ser Thr Leu Tyr Thr Val 430 435 440 ccc ttt aac ctc att act gag tac cac cgc gag gaa gaa aag gag agg 1396 Pro Phe Asn Leu Ile Thr Glu Tyr His Arg Glu Glu Glu Lys Glu Arg 445 450 455 cag cag gcc cca gga ggg gac cca gac aac agc gtg aga ggg aag ggc 1444 Gln Gln Ala Pro Gly Gly Asp Pro Asp Asn Ser Val Arg Gly Lys Gly 460 465 470 atg gac tgc gcc acc ctc aca tgc atg gtg cag ctg gct cag atc ctg 1492 Met Asp Cys Ala Thr Leu Thr Cys Met Val Gln Leu Ala Gln Ile Leu 475 480 485 gtc gga ggt ggc ctg ggc ttt ctg gtc aac aca gcc ggg acc gtt gtc 1540 Val Gly Gly Gly Leu Gly Phe Leu Val Asn Thr Ala Gly Thr Val Val 490 495 500 505 gtc gtg gtg atc aca gcg tct gcg gtg gca ctg ata ggc tgt tgc ttt 1588 Val Val Val Ile Thr Ala Ser Ala Val Ala Leu Ile Gly Cys Cys Phe 510 515 520 gtc gct ctc ttt gtt aga tat gtg gat taggtcaata aagagacaat 1635 Val Ala Leu Phe Val Arg Tyr Val Asp 525 530 gaccctaaaa aaaaa 1650 4 530 PRT Homo sapiens 4 Met Gly Ser Asn Ser Gly Gln Ala Gly Arg His Ile Tyr Lys Ser Leu 1 5 10 15 Ala Asp Asp Gly Pro Phe Asp Ser Val Glu Pro Pro Lys Arg Pro Thr 20 25 30 Ser Arg Leu Ile Met His Ser Met Ala Met Phe Gly Arg Glu Phe Cys 35 40 45 Tyr Ala Val Glu Ala Ala Tyr Val Thr Pro Val Leu Leu Ser Val Gly 50 55 60 Leu Pro Ser Ser Leu Tyr Ser Ile Val Trp Phe Leu Ser Pro Ile Leu 65 70 75 80 Gly Phe Leu Leu Gln Pro Val Val Gly Ser Ala Ser Asp His Cys Arg 85 90 95 Ser Arg Trp Gly Arg Arg Arg Pro Tyr Ile Leu Thr Leu Gly Val Met 100 105 110 Met Leu Val Gly Met Ala Leu Tyr Leu Asn Gly Ala Thr Val Val Ala 115 120 125 Ala Leu Ile Ala Asn Pro Arg Arg Lys Leu Val Trp Ala Ile Ser Val 130 135 140 Thr Met Ile Gly Val Val Leu Phe Asp Phe Ala Ala Asp Phe Ile Asp 145 150 155 160 Gly Pro Ile Lys Ala Tyr Leu Phe Asp Val Cys Ser His Gln Asp Lys 165 170 175 Glu Lys Gly Leu His Tyr His Ala Leu Phe Thr Gly Phe Gly Gly Ala 180 185 190 Leu Gly Tyr Leu Leu Gly Ala Ile Asp Trp Ala His Leu Glu Leu Gly 195 200 205 Arg Leu Leu Gly Thr Glu Phe Gln Val Met Phe Phe Phe Ser Ala Leu 210 215 220 Val Leu Thr Leu Cys Phe Thr Val His Leu Cys Ser Ile Ser Glu Ala 225 230 235 240 Pro Leu Thr Glu Val Ala Lys Gly Ile Pro Pro Gln Gln Thr Pro Gln 245 250 255 Asp Pro Pro Leu Ser Ser Asp Gly Met Tyr Glu Tyr Gly Ser Ile Glu 260 265 270 Lys Val Lys Asn Gly Tyr Val Asn Pro Glu Leu Ala Met Gln Gly Ala 275 280 285 Lys Asn Lys Asn His Ala Glu Gln Thr Arg Arg Ala Met Thr Leu Lys 290 295 300 Ser Leu Leu Arg Ala Leu Val Asn Met Pro Pro His Tyr Arg Tyr Leu 305 310 315 320 Cys Ile Ser His Leu Ile Gly Trp Thr Ala Phe Leu Ser Asn Met Leu 325 330 335 Phe Phe Thr Asp Phe Met Gly Gln Ile Val Tyr Arg Gly Asp Pro Tyr 340 345 350 Ser Ala His Asn Ser Thr Glu Phe Leu Ile Tyr Glu Arg Gly Val Glu 355 360 365 Val Gly Cys Trp Gly Phe Cys Ile Asn Ser Val Phe Ser Ser Leu Tyr 370 375 380 Ser Tyr Phe Gln Lys Val Leu Val Ser Tyr Ile Gly Leu Lys Gly Leu 385 390 395 400 Tyr Phe Thr Gly Tyr Leu Leu Phe Gly Leu Gly Thr Gly Phe Ile Gly 405 410 415 Leu Phe Pro Asn Val Tyr Ser Thr Leu Val Leu Cys Ser Leu Phe Gly 420 425 430 Val Met Ser Ser Thr Leu Tyr Thr Val Pro Phe Asn Leu Ile Thr Glu 435 440 445 Tyr His Arg Glu Glu Glu Lys Glu Arg Gln Gln Ala Pro Gly Gly Asp 450 455 460 Pro Asp Asn Ser Val Arg Gly Lys Gly Met Asp Cys Ala Thr Leu Thr 465 470 475 480 Cys Met Val Gln Leu Ala Gln Ile Leu Val Gly Gly Gly Leu Gly Phe 485 490 495 Leu Val Asn Thr Ala Gly Thr Val Val Val Val Val Ile Thr Ala Ser 500 505 510 Ala Val Ala Leu Ile Gly Cys Cys Phe Val Ala Leu Phe Val Arg Tyr 515 520 525 Val Asp 530 5 1986 DNA mouse CDS (5)..(1594) 5 ggtc atg agt gga agc aat ggg ccg act gac acc cat acc tat caa tcc 49 Met Ser Gly Ser Asn Gly Pro Thr Asp Thr His Thr Tyr Gln Ser 1 5 10 15 tta gcc gag gat tgc ccc ttt ggc tct gtg gag caa ccc aag aga tcc 97 Leu Ala Glu Asp Cys Pro Phe Gly Ser Val Glu Gln Pro Lys Arg Ser 20 25 30 aca ggg aga ctt gtc atg cac agc atg gcc atg ttt ggc cga gag ttt 145 Thr Gly Arg Leu Val Met His Ser Met Ala Met Phe Gly Arg Glu Phe 35 40 45 tgc tat gcg gtg gag gca gct tat gtg act cca gtt ctg ctc agc gtg 193 Cys Tyr Ala Val Glu Ala Ala Tyr Val Thr Pro Val Leu Leu Ser Val 50 55 60 ggc ctg cct aag agc ctg tac agc atg gtg tgg ctc cta agc ccc atc 241 Gly Leu Pro Lys Ser Leu Tyr Ser Met Val Trp Leu Leu Ser Pro Ile 65 70 75 ttg gga ttc ctg ctc cag cct gtg gtg gga tca gcc agt gat cac tgc 289 Leu Gly Phe Leu Leu Gln Pro Val Val Gly Ser Ala Ser Asp His Cys 80 85 90 95 agg gcc cgt tgg ggt cgc cgg aga cca tac atc ctg act ctg gcc att 337 Arg Ala Arg Trp Gly Arg Arg Arg Pro Tyr Ile Leu Thr Leu Ala Ile 100 105 110 atg atg ctc ttg gga atg gct ctg tac ctc aat gga gat gcg gtc gta 385 Met Met Leu Leu Gly Met Ala Leu Tyr Leu Asn Gly Asp Ala Val Val 115 120 125 tca gct ttg gtt gct aac cca agg cag aag ctg atc tgg gcc ata agc 433 Ser Ala Leu Val Ala Asn Pro Arg Gln Lys Leu Ile Trp Ala Ile Ser 130 135 140 atc acc atg gta ggt gtg gtt ctc ttc gat ttt tct gct gac ttc att 481 Ile Thr Met Val Gly Val Val Leu Phe Asp Phe Ser Ala Asp Phe Ile 145 150 155 gac ggg ccc atc aaa gcc tac tta ttt gat gtc tgc tcc cac cag gac 529 Asp Gly Pro Ile Lys Ala Tyr Leu Phe Asp Val Cys Ser His Gln Asp 160 165 170 175 aag gag aag ggc ctc cac tac cat gcc ctc ttc aca ggt ttt gga ggt 577 Lys Glu Lys Gly Leu His Tyr His Ala Leu Phe Thr Gly Phe Gly Gly 180 185 190 gcc ctt ggc tac att ttg ggt gcc ata gac tgg gtg cat cta gat ctg 625 Ala Leu Gly Tyr Ile Leu Gly Ala Ile Asp Trp Val His Leu Asp Leu 195 200 205 gga agg ctg ctg ggc aca gaa ttc cag gtc atg ttc ttc ttc tct gcc 673 Gly Arg Leu Leu Gly Thr Glu Phe Gln Val Met Phe Phe Phe Ser Ala 210 215 220 ctg gtt ctc atc ttg tgc ttc atc aca cac ctg tgc agt atc cct gaa 721 Leu Val Leu Ile Leu Cys Phe Ile Thr His Leu Cys Ser Ile Pro Glu 225 230 235 gct cca ctc aga gat gct gca act gac cct ccc tca cag cag gac cct 769 Ala Pro Leu Arg Asp Ala Ala Thr Asp Pro Pro Ser Gln Gln Asp Pro 240 245 250 255 cag ggc tcg tcg ctg tca gcc agt ggg atg cat gaa tac ggt tct att 817 Gln Gly Ser Ser Leu Ser Ala Ser Gly Met His Glu Tyr Gly Ser Ile 260 265 270 gag aaa gtt aaa aat gga ggt gca gac aca gag cag cca gta cag gaa 865 Glu Lys Val Lys Asn Gly Gly Ala Asp Thr Glu Gln Pro Val Gln Glu 275 280 285 tgg aaa aac aaa aag cct tct ggc cag agt cag agg aca atg tcg atg 913 Trp Lys Asn Lys Lys Pro Ser Gly Gln Ser Gln Arg Thr Met Ser Met 290 295 300 aag tca ctc ctt cgg gca tta gta aac atg cct tcc cat tat cgc tgc 961 Lys Ser Leu Leu Arg Ala Leu Val Asn Met Pro Ser His Tyr Arg Cys 305 310 315 ctt tgc gtc agc cac ctg att gga tgg act gcc ttc ctg tca aac atg 1009 Leu Cys Val Ser His Leu Ile Gly Trp Thr Ala Phe Leu Ser Asn Met 320 325 330 335 ctc ttc ttc aca gat ttc atg gga cag att gta tac cat ggg gat ccc 1057 Leu Phe Phe Thr Asp Phe Met Gly Gln Ile Val Tyr His Gly Asp Pro 340 345 350 tac ggt gca cac aac tcc acg gag ttt ctc atc tat gaa aga gga gtt 1105 Tyr Gly Ala His Asn Ser Thr Glu Phe Leu Ile Tyr Glu Arg Gly Val 355 360 365 gag gtc gga tgt tgg ggc ttg tgc atc aac tct gtg ttt tct tca gtt 1153 Glu Val Gly Cys Trp Gly Leu Cys Ile Asn Ser Val Phe Ser Ser Val 370 375 380 tat tca tac ttt cag aaa gct atg gtc tcc tac att gga tta aaa ggc 1201 Tyr Ser Tyr Phe Gln Lys Ala Met Val Ser Tyr Ile Gly Leu Lys Gly 385 390 395 ctt tat ttc atg gga tat ttg ctc ttt ggc ctg gga aca gga ttc ata 1249 Leu Tyr Phe Met Gly Tyr Leu Leu Phe Gly Leu Gly Thr Gly Phe Ile 400 405 410 415 gga ctc ttt cca aat gtg tac tct act ctg gtc ctc tgt tct atg ttt 1297 Gly Leu Phe Pro Asn Val Tyr Ser Thr Leu Val Leu Cys Ser Met Phe 420 425 430 ggt gta atg tcc agc aca ttg tac act gtg ccc ttt aac ctc att gct 1345 Gly Val Met Ser Ser Thr Leu Tyr Thr Val Pro Phe Asn Leu Ile Ala 435 440 445 gag tac cac cgt gaa gag gag aaa gag aag ggg cag gaa gcg cca gga 1393 Glu Tyr His Arg Glu Glu Glu Lys Glu Lys Gly Gln Glu Ala Pro Gly 450 455 460 ggc cct gac aac cag ggg aga ggc aaa ggc gtg gac tgc gct gct ctc 1441 Gly Pro Asp Asn Gln Gly Arg Gly Lys Gly Val Asp Cys Ala Ala Leu 465 470 475 acc tgc atg gta cag ctg gct cag atc ttg gtt gga ggt ggc ctg ggc 1489 Thr Cys Met Val Gln Leu Ala Gln Ile Leu Val Gly Gly Gly Leu Gly 480 485 490 495 ttc ctg gtc aac atg gct ggg agt gtc gtc gtc gtg gtg atc aca gcg 1537 Phe Leu Val Asn Met Ala Gly Ser Val Val Val Val Val Ile Thr Ala 500 505 510 tct gcg gtg tcc ctg ata ggc tgt tgc ttt gtg gcg ctc ttc gta aga 1585 Ser Ala Val Ser Leu Ile Gly Cys Cys Phe Val Ala Leu Phe Val Arg 515 520 525 tat gta gat taggttcata aaaagacacc caccaaacct cagwcaaaag 1634 Tyr Val Asp 530 aagcccatgg caagaaaccg ctcttttctc ggactcttcg gtgttgttgg ggactgtatg 1694 cctcatgctg gcactgcgcc ttgcacatac atgtggggaa ggtgtggtgg gattgcccgc 1754 tcccctgcat cttccttact gccccgcaat gcaggatgag agagtcaagt tctgcttctc 1814 aagagaattt caagcctcca tcatgtctta ggaacaaatg catcttacyt gtaatgctta 1874 taaataattt ttaaaagtca attcatatac cactgggaaa tcacacattt catcaaattc 1934 agaagcactt aaaacatgac cgagtgtttt ttttaaaaaa aaaaaaaaaa aa 1986 6 530 PRT mouse 6 Met Ser Gly Ser Asn Gly Pro Thr Asp Thr His Thr Tyr Gln Ser Leu 1 5 10 15 Ala Glu Asp Cys Pro Phe Gly Ser Val Glu Gln Pro Lys Arg Ser Thr 20 25 30 Gly Arg Leu Val Met His Ser Met Ala Met Phe Gly Arg Glu Phe Cys 35 40 45 Tyr Ala Val Glu Ala Ala Tyr Val Thr Pro Val Leu Leu Ser Val Gly 50 55 60 Leu Pro Lys Ser Leu Tyr Ser Met Val Trp Leu Leu Ser Pro Ile Leu 65 70 75 80 Gly Phe Leu Leu Gln Pro Val Val Gly Ser Ala Ser Asp His Cys Arg 85 90 95 Ala Arg Trp Gly Arg Arg Arg Pro Tyr Ile Leu Thr Leu Ala Ile Met 100 105 110 Met Leu Leu Gly Met Ala Leu Tyr Leu Asn Gly Asp Ala Val Val Ser 115 120 125 Ala Leu Val Ala Asn Pro Arg Gln Lys Leu Ile Trp Ala Ile Ser Ile 130 135 140 Thr Met Val Gly Val Val Leu Phe Asp Phe Ser Ala Asp Phe Ile Asp 145 150 155 160 Gly Pro Ile Lys Ala Tyr Leu Phe Asp Val Cys Ser His Gln Asp Lys 165 170 175 Glu Lys Gly Leu His Tyr His Ala Leu Phe Thr Gly Phe Gly Gly Ala 180 185 190 Leu Gly Tyr Ile Leu Gly Ala Ile Asp Trp Val His Leu Asp Leu Gly 195 200 205 Arg Leu Leu Gly Thr Glu Phe Gln Val Met Phe Phe Phe Ser Ala Leu 210 215 220 Val Leu Ile Leu Cys Phe Ile Thr His Leu Cys Ser Ile Pro Glu Ala 225 230 235 240 Pro Leu Arg Asp Ala Ala Thr Asp Pro Pro Ser Gln Gln Asp Pro Gln 245 250 255 Gly Ser Ser Leu Ser Ala Ser Gly Met His Glu Tyr Gly Ser Ile Glu 260 265 270 Lys Val Lys Asn Gly Gly Ala Asp Thr Glu Gln Pro Val Gln Glu Trp 275 280 285 Lys Asn Lys Lys Pro Ser Gly Gln Ser Gln Arg Thr Met Ser Met Lys 290 295 300 Ser Leu Leu Arg Ala Leu Val Asn Met Pro Ser His Tyr Arg Cys Leu 305 310 315 320 Cys Val Ser His Leu Ile Gly Trp Thr Ala Phe Leu Ser Asn Met Leu 325 330 335 Phe Phe Thr Asp Phe Met Gly Gln Ile Val Tyr His Gly Asp Pro Tyr 340 345 350 Gly Ala His Asn Ser Thr Glu Phe Leu Ile Tyr Glu Arg Gly Val Glu 355 360 365 Val Gly Cys Trp Gly Leu Cys Ile Asn Ser Val Phe Ser Ser Val Tyr 370 375 380 Ser Tyr Phe Gln Lys Ala Met Val Ser Tyr Ile Gly Leu Lys Gly Leu 385 390 395 400 Tyr Phe Met Gly Tyr Leu Leu Phe Gly Leu Gly Thr Gly Phe Ile Gly 405 410 415 Leu Phe Pro Asn Val Tyr Ser Thr Leu Val Leu Cys Ser Met Phe Gly 420 425 430 Val Met Ser Ser Thr Leu Tyr Thr Val Pro Phe Asn Leu Ile Ala Glu 435 440 445 Tyr His Arg Glu Glu Glu Lys Glu Lys Gly Gln Glu Ala Pro Gly Gly 450 455 460 Pro Asp Asn Gln Gly Arg Gly Lys Gly Val Asp Cys Ala Ala Leu Thr 465 470 475 480 Cys Met Val Gln Leu Ala Gln Ile Leu Val Gly Gly Gly Leu Gly Phe 485 490 495 Leu Val Asn Met Ala Gly Ser Val Val Val Val Val Ile Thr Ala Ser 500 505 510 Ala Val Ser Leu Ile Gly Cys Cys Phe Val Ala Leu Phe Val Arg Tyr 515 520 525 Val Asp 530 

1. A nucleic acid encoding medaka protein B or homologues thereof.
 2. A nucleic acid listed in Seq. No. 1 that encodes medaka protein B.
 3. A nucleic acid listed in Seq. No. 3 that encodes mouse protein B/AIM-1.
 4. A method of suppressing melanin production characterized in that expression of at least one nucleic acid according to any one of claims 1-3 is suppressed. 